Chromosoma(1992) 102:S133-S141

CHROMOSOMA 9 Springer-Verlag1992

DNA polymerase 5 and holoenzymes from calf thymus Vladimir Podust, Victor Mikhailov*, Anthi Georgaki, and Ulrich Hiibscher Departmentof Pharmacologyand Biochemistry,UniversityZiidch-Irchel,Winterthurerstrasse190,CH-8057Ztirich,Switzerland ReceivedSeptember12, 1992

Abstract. Replication of singly-DNA primed M13 DNA by DNA polymerase (pol) ~ completely relies on the simultaneous addition of proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) (or E.coli singlestrand DNA binding protein, SSB). P o l e core alone is able to synthesize the products on singly-primed ssDNA. However, DNA synthesis by pol Iz was stimulated up to 10-fold upon addition of the three auxiliary proteins PCNA, RF-C and SSB. This stimulation of pol E by PCNA/RF-C/SSB appears to be the superposition of two events: pol e holoenzyme (pol ~, PCNA, RF-C) synthesized longer products than its p o l e core counterpart, but elongated less primers. Furthermore, we analyzed the cooperative action of pol a/primase with pol 5 or pol E holoenzymes on unprimed M13 DNA. While pol ~ displayed higher dNMP incorporation than pol ~., when a single primer was preannealed to DNA, p o l ~ was more efficient in the utilization of the primers synthesized by pol a/primase. Under these conditions both longer products and a higher amount of dNMP incorporation was found for p o l e holoenzyme, than for pol 5. Our data support the hypothesis of pol 5 as the leading and pol E as the second lagging strand replication enzyme. * OnsabbaticalleavefromKol'tzovInstituteofDevelopmentalBiology, 117334,Moscow,Russia Abbreviations: pol,DNApolymerasc;PCNA,proliferatingcellnuclear antigen;R_P-A,replicationproteinA; R.F-C,replicationfactorC; SSB, single-strandDNAbindingprotein;ssDNA,single-strandDNA;SV40, simianvirus40; T antigen,SV40 largetumorantigen;DTF,dithiothrcitol;BSA,bovineserumalbumin Correspondence to: U. Hiibscher

Introduction Replicative pol's from bacteriophages to man all appear to exist at several levels of complexity starting from the catalytic subunit to a structural form that resembles the in vivo structure (Komberg and Baker 1992). The bacteriophage T4 encoded pol, for instance, consists of a single 109 kDa polypeptide and displays only moderatly processive DNA synthesis. When associated with the auxiliary proteins gp44/gp62 and gp45, however T4-pol becomes a highly processive enzyme (Alberts 1987). In E.coli three pol's have been identified (named pol I, II and III) and of these, pol III has been determined to be the replicative enzyme. The catalytic subunit of pol III (txsubunit) is associated with the e-subunit (3'-5' proofreading exonuclease) and the 0-subunit forming the core enzyme (see McHenry 1991 and references therein for details). The next level of complexity is pol III', consisting ofa dimer ofpol III cores and x-subunits. Finally, the pol III auxiliary proteins (13--subunit, different subassemblies of T-complexes) form with the pol III' an asymmetrical dimer of 22 polypeptides which has the properties for a true replicative holoenzyme (e.g. processivity and recycling capacity). This has been called pol III holoenzyme (Maki et al. 1988). In eukaryotes at least five different pol's have been identified (a, I~, Y, ~5and e, Burgers et al. 1990). Recent data suggest that three of them, namely pol's a, ~ and e, are involved in DNA replication (reviewed in Thtmmes and Hiibscher 1990, Wang 1991, H/ibscher and ThSmmes 1992). Replication studies of SV40 DNA indicate that several additional factors such as PCNA (Prelich et al. 1987), RF-C (Tsurimoto and Stillman 1989a) and RP-A (Wold and Kelly 1988) are required for an efficient in vitro replication activity. These factors appear to be es-

S134 sential replication proteins and have been categorized as DNA polymerase accessory proteins (Tsurimoto and Stillman 1990). PCNA's from different sources are structurally and functionally conserved. Yeast pol ~i can be stimulated by bovine PCNA and vice versa (Burgers 1988). Physical and functional homologies as well as structural similarities suggest that PCNA from eukaryotes is analogous to the gene 45 protein from bacteriophage T4 and to the [3-subunit of pol III holoenzyme (Tsurimoto and Stillman 1990). RPA represents the eukaryotic counterpart of T4 gp32 and E.coli SSB. However, this protein is composed of three tightly associated subunits and seems to be conserved from yeast to man (see e.g. references in Kim et al. 1992). RP-A acts in several steps of DNA replication. It can be replaced by other SSB's in the T antigen-dependent unwinding of SV40 DNA (Kenny et al. 1989) as well as in DNA synthesis on the primed ssDNA by pol ~i and p o l e in the presence of PCNA and RF-C (Lee et al. 1991b, Yoder and Burgers 1991, and see below). However, other SSB's could not substitute for the human RP-A in SV40 DNA replication in vitro (Kenny et al. 1989) possibly due to pol r dependent synthesis of Okazaki fragments. Indeed, a highly specific interaction of T antigen, RP-A and pol ct/primase has been demonstrated (Dornreiter et al. 1992). Finally, the 32 kDa subunit of RP-A is phosphorylated in a cell cycle-dependent manner and this phosphorylation event affects DNA replication (Dutta and Stillman 1992). These data suggest that RP-A not only stabilizes the unwound duplex and prevents the nonspecific interaction of various proteins with ssDNA, but also specifically interacts with components of the replication machinery. The third essential auxiliary protein, named RF-C, was first isolated from human cells using its ability to support SV40 DNA replication in vitro (Tsurimoto and Stillman 1989a). Later, using a complementation assay that enabled pol 5 to replicate a singly-primed DNA in the presence of PCNA and RP-A or SSB, a multisubunit complex called RF-C was isolated from Hela cells (Lee et al. 1991a), yeast (Yoder and Burgers 1991, Fien and Stillman 1992) and calf thymus (Podust et al. 1992). All RF-C preparations contain a high molecular weight polypeptide (110-155 kDa) and a set of smaller polypeptides (36--42 kDa). RF-C displays DNA binding activity and the high M r subunit was shown to be responsible for this interaction (Tsurimoto and Stillman 1991a). The data on the specificity of the RF-C/DNA interaction are variable and depend on the experimental method used. Using a nitro-

cellulose filter binding assay, RF-C was shown to bind to the template-primer complex, but not to the single-strand template alone (Tsurimoto and Stillman 1990, Lee et al. 1991a). By using a gel-shift assay, however, RF-C complexed with ssDNA and even with fully dsDNA (Tsurimoto and Stillman 1991a). But again, RF-C displayed the highest affinity for template-primer DNA. RFC binds to the template-primer junction and ATP increase this specific binding. Footprinting experiments showed that RF-C covers 20 nucleotides of template and 15 nucleotides of primer (Tsurimoto and Stillman 1991a). In the presence of ATP, PCNA formed a stable primer recognition complex consisting of DNA/RF-C/PCNA which could be detected by gel-shift and footprinting assays (Tsurimoto and Stillman 1991a) as well as by gel filtration (Lee and Hurwitz 1990, Burgers 1991, Podust et al. 1992). Biochemical studies indicate that RF-C is the functional analog of the bacteriophage T4 gp44/gp62 complex and the E.coli T-complex ofpol III holoenzyme. Finally, the aminoacid sequences of the 37 kDa and 40 kDa subunits of human RF-C showed a substantial homology with T4 gp44 and to a lesser extent with the xand y-subunits ofE.coli pol III holoenzyme (Chen et al. 1992a, b). In calf thymus, pol ~ and pole can form similar structures in the presence of the auxiliary proteins PCNA, RFC and SSB, which have been termed holoenzymes (Podust et al. 1992). In the following, we have compared these two holoenzymes in their capacity to elongate both preexisting DNA primers and RNA-DNA primers synthesized by pol ~primase.

Materials and methods Enzymes, Proteins and Nucleic Acids: Calfthyrnus enzymes and proteins were isolated as described: pole (Focher et al. 1989), pol 8 (Weiser et al. 1991), PCNA (Prelich et al. 1987), RF-C (Podust et al. 1992), RPA (Georgaki et al. 1992). E. coli SSB was purified from the overproducer RLM 727 (gift ofMartine Defais, CNRS, Toulouse) according to Lohman et al. (1986). Pol t~/primase was immunoalTmity purified in a two-step procedure. The crude extract was chromatographed on phosphocellulose according to Nasheuer and Grosse (1987). The eluate was dialyzed against 30 mM potassium phosphate (pH 7.4), 40 mM NaC1, 10% (v/v) glycerol and loaded on a 1 ml immunoaffmity column. The later was prepared by covalent linking of anti-calfthymus pol ct antibody 17 (kind gift of Dr. L.M.S. Chang) to Tresyl-Sepharose (Pharmacia) according to manufacturer protocol. The column was washed with 50 mM Tris-HC1 (pH 8.0), 150 mM NaC1 and eluted with 3.2 M MgC12 buffered with 50 mM Tris-HC1 (pH 8.5) (Chang et al. 1984). Active eluate fractions were pooled and dialyzed against 50 mM Tris-HCl (pH 7.5), 30 mM NaC1, 4 mM DTI', 10% (v/v) glycerol. Single-strand M13(mp11) DNA was prepared according to Sambrook et al. (1989). Two different oligonucleotides were used to prepare singlyprimed M13 DNA: XbaI-EcoRI polylinker oligo 9013690 (30-met) or an oligonucleotide complementary to 7041-7080 nt region o f M 13

S135 genome(40-mer). Both oligonuclcotideswereannealedto M13 DNA as described (Podust et al. 1992).

Replication assays: 1) Singly-primedM13 DNA: a final volume of 25 l.tlcontained the following components:40 mM Tris-HC1(pH 7.5), 1 mM D'I'T,0.2 mg/ml BSA, 1 mM ATP, 10 mM MgC12,dATP, dGTP, dCTP each at 50 btM, 15 I.tMof [3H]dTI'P(1500 cpm/pmol)(or dATP, dGTP, d'ITP each at 50 paM,15 IxlVIof [32p]dCTP(12,000 cpm/pmol)), 100ng ofsingly-DNAprimed M13 DNA, 100 ng ofPCNA, 6 rig of RFC, 350 ng ofE.coli SSB or 600 ng of RP-A and 0.25 U of either pol 8 or pol e. In some experimentsthe primerwas 5'-[32P]-phosphorylated, dNTP's were then used unlabelled at 50 I.tMeach. 2) UnprimedM13 DNA: a final volume of 25 Isl containedthe followingcomponents:40 mM Tris-HCl (pH 7.5), 1 mM DTr, 0.2 mg/ml BSA, 10 mM MgC12, GTP, CTP, UTP each at 0.1 mM, 1mM ATP, dATP,dGTP,dCTPeach at 50 ~,I, 15 }aMof [32p]dCTP(12,000 epm/pmol),200 ng ofunprimed M13 DNA, 100 ng ofPCNA, 6 ng of RF-C, 600 ng ofE.coli SSB or 1200 ng of RP-A, 0.25-0.4 U ofpol otand 0.25 U of either pol 8 or pol e. 3) Gapped-DNA:a final volume of 25 I.tlcontainedthe samecomponentsas for singly-primedMI 3 DNA assay.GappedDNA was prepared by annealingfirsta 40-mer primer ([32p]-phoshorylatedbeforeannealing, 2-molarexcessto M 13 DNA) and seconda 40-mer oligonucleotide complementaryto a regiondownstream( 10-molarexcessto M13 DNA) thus leaving a 230 nucleotides sap (Podust and Hiibscher,submitted). dNTP's were used unlabelled at 50 I.tMeach. Productana!ysis. Reactionmixtttreswere incubatedat 37~ and terminated first by chilling and second by addition of 2.3 I.tlof a mixture containing 10% (w/v) SDS and 0.1 M EDTA (pH 8.0). DNA was precipitated with ethanol as routine (Sarnbrook et al. 1989). Alkaline agaroseeleclrophoresiswas performedon 1-1.5% agarosegelsat 2-3 V/ cm at room temperature in a solution containing 30 mM NaOH and 2 mM EDTA. ARer electrophoresis,the gels were neutralized in 0.6 M Tris-HC1(pH 7.5), stainedwith ethidiumbromide,photographed,dried on DE-81 paper and exposed to X-ray films. Results and discussion

Pol ~ and pol ~ are both dependent on the auxiliary proteins PCNA, RF-C and RP-A (SSB) for efficient DNA synthesis on singly-primed DNA, but in a different way RF-C alone has been reported to stimulate pol ~ and pol on primed M13 DNA (Tsurimoto and Stillman 1989b). However, Lee et al. (1991a) showed that pol (z activity was unaffected by PCNA, RF-C or a combination of these two proteins. We also could not detect any stimulatory effect of calf thymus PCNA and RF-C on the bovine pol ~ activity (Podust and Hfibscher, unpublished observation). Formation of a primer recognition complex on primed DNA alters the catalytical properties of pol ~, and e. Pol o~was reported to be strongly inhibited when PCNA, RF-C and RP-A were present in combination in the reaction mixture (Tsurimoto and Stillman 1991b). In contrast, pol ~ absolutely required the presence of all three auxiliary factors (PCNA, RF-C, RP-A) in order to elongate singly-primed DNA (Tsurimoto and Stillman 1989b, Lee etal. 1991a).

Figure 1 shows the absolute dependence of bovine pol 5 on these three auxiliary proteins. The dependence on ATP was not complete since other dNTP's present in the reaction mixture could partially substitute for ATP (Lee et al. 1991a). The origin of SSB does not seem to be essential for pol ~ holoenzyme synthesis. Both E.coli SSB and bovine RP-A were similarily efficient in the stimulation of DNA synthesis by the bovine pol ~ holoenzyme (Fig. 3, lanes 2, 3, 8, 9, see also Lee et al. (1991b) for human pol 5 holoenzyme). The PCNA/RF-C complex is known to have significant affinity for ssDNA (Tsurimoto and Stillman 1991a) and in the absence of SSB is unproductively distributed on the abundant ss-region of the substrate. Thus, the main function of SSB in this system appears to reduce non-specific DNA binding by PCNA/RF-C. In contrast, p o l e seems to interact with the auxiliary proteins in a more complex way. The core pol E has been reported to utilize singly-primed ssDNA as a substrate. In the presence of E.coli SSB, yeast p o l e efficiently elongated primers to the full length of substrate and no influence of PCNA, RF-C or PCNA/RF-C could be detected. However, at increased salt concentrations (85-100 m M NaCI) when core p o l e is strongly inhibited, addition of primer recognition complex resulted in recovery of polymerization, suggesting that a p o l e holoenzyme was formed (Yoder and Burgers 1991, Lee et al. 1991b). In contrast to yeast, the human and bovine core pol ~ poorly elongated primed ssDNA, yielding only short products. Addition of a combination of PCNA, RF-C and SSB stimulated DNA synthesis up to 10-fold (Lee et al. 1991b, Podust et al. 1992, see also Fig. 1). We find that while bovine RP-A and E.r SSB were equally effective in

Fig. 1. Replication of singly-DNA primed M13 DNA is dependenton PCNA, RF-C and SSB. Replication of singly-DNA primed (30-mer) M13 DNA was carried out as described in Materials and methods. Mixtures were incubated for 30 min and the acid-insoluble products analyzed

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Fig. 2. Comparison of calf thymus DNA polymerase 8 and e holoenzymes in their ability to utilize decreasing amounts o/'3'-0H termini. SIngly-DNA primed (40-mer) M13 DNA was diluted with unprimed M13 DNA thus decreasing the concentration of 3'-OH termini at the fixed concentration of MI 3 DNA. Replication reactions with all necessary components (see Fig. 1) were carried out as described in Materials and methods. The data were analyzed in [S]/V versus [S] coordinates (Hanes-Woolf plot), where interseption of curve with X-axis corresponds to K m value

stimulation ofpol 8 holoenzyme formation, bovine RP-A was much less effective than the E.coli counterpart in pol holoenzyme formation, yielding only about 30% of the level of DNA synthesis. A similar observation, namely that E.coli SSB can more effectively substitute for the eukaryotic counterpart, was also reported with pol holoenzymes from yeast (Yoder and Burgers 1991) and Hela cells (Lee et al. 1991b).

(Fig. 3, see also Lee et al. 1991b, Podust et al. 1992). We compared synthesis on M13 DNA by pole to pol ~i,when either 5'-labelled primer and unlabelled dNTP's or unlabelled primer and labelled dNTP's were used. In the former case, both the initial substrate and the products of all lengths were equally labelled. Pole holoenzyme synthesized longer products in comparison to pole core (Fig. 3, compare lanes 6 and 12 versus 4 and 10), but notably less primers were elongated under the same conditions (Fig. 3, lane 6 versus 4). To better characterize these data, we used a defined gapped DNA which can serve as a model for Okazaki fragment gap-filling (see Materials and methods). Pol e core, p o l e holoenzyme and pol ~5 holoenzyme (for pol fi data not shown) were able to fill the gap completely, however strong pausing sites inside the gap were detected for pole core (Fig. 4). The concentration of E.coli SSB was varied while that of PCNA and RF-C, if present, was constant. Thus, higher concentrations of SSB favoured the formation of a productive recognition complex. SSB strongly inhibited filling of the gap in DNA by pol e in the absence of PCNA/RF-C (Fig. 4, C, right autoradiogram below). Low levels of SSB (0-200 ng per 100 ng of DNA) were not sufficient to sup-

Pol e holoenzyme synthesizes longer products but elongates less primers than pol ~ core The primer recognition complex mediates the binding of pol 6 to the 3'-OH end of a primer, thereby increasing the apparent affinity of this enzyme for the substrate (Lee et al. 1991a). Since PCNA/RF-C appears to stimulate pol 8 and pol e on SSB-eovered, singly-primed DNA in a similar manner, that is formation of a holoenzyme complex, one would expect that the mechanism of this stimulation would be the same. In order to assess this hypothesis, we have compared the affinities ofpol's 6 and e for the preformed primer recognition complex in experiments where the activity of both pol's was measured at gradually decreasing primer concentration (Fig. 2). The Km value of pole for primers was 1.2 nM (concentration of 3'-ends of the primers), which was more than 2.5-fold lower than the Km value of pol 8 (3.2 nM). The primer recognition complex increased the total incorporation of labelled dNMP into DNA by human and bovine pol~ up to 10-fold and increased the length of synthetic products

Fig. 3. Product analysis of calf thymus DNA polymerase 5 and e holoenzymes in the presence of RP-A or E.coli SSB. Replication of singly-DNA primed (40-mer) M13 DNA was carried out in the presence of PCNA (100 ng), RF-C (6 ng) and ATP (1 mM) as described in Experimental Procedures. Other added components are indicated on top of each lane. Mixtures were incubated for 30 rnin and analyzed on a 1.5% alkaline agarose gel. Primer oligonucleotide for annealing was taken in 2-fold molar excess to the template. Lanes I-6: primer was 5'-[32p]-phosphorylatedbefore annealing and unlabeled dNTP's were used for the reaction. Lanes 7-12: primer was unphosphorylated, the product was labelled with [32p]dCTP in the presence of the other dNTP's as described in Materials and methods

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port the productive binding of PCNA/RF-C to Y-OH termini and inhibited pole in the presence of PCNA and RFC. In contrast, high levels of SSB (300-400 ng) provided optimal conditions for DNA synthesis by both pol 8 and p o l e (Fig. 4, A, B) and at the same time restored the number of primers elongated up to the full length of the gap, but not to the same level as in the absence of SSB. (Fig. 4, C, left autoradiogram below). Therefore the total stimulation of pol e synthesis by PCNA/RF-C/SSB appears to be the superposition of two processes: p o l e holoenzyme synthesized on one hand longer products, which might be due to an easier overcoming of the sec-

Fig. 4. Dependence of DNA polymerase 8 and e holoenzymes synthesis on the concentration of SSB. Replicationreactionswerecarried out underoptimalconditionsas describedin Materialsand methodsby using singly-DNAprimed M13 DNA (Panels A and B) or gapped M13 DNA (PanelsC, D and the autoradiogramsbelow).PanelsA and B: Replicationof singly-DNAprimed(40-mer) M13 DNA was carried out as describedin Materialsand methodsand the dependencyon added E.coli SSB determinedfor pol E(A) and pol 8 (B) holoenzymes. Panels C andD: The number of primers elongatedwas analyzedon gapped templateas describedin Materials and methodsand the dependencyof addedE.coli SSB determinedfor pol E(C) and pol 5 (D) holoenzymes.Notethat for pole the numberof primers elongatedwas also determinedfor pol E core (dotted line, no PCNA/RF-C). The productswere separatedon 5% PAGEcontaining7 M ureaand quanrifled as outlined in the autoradiograrnsbelow. Autoradiogramsof products synthesizedby pol Eholoenzyme(left)and pole core (fight). The products of the completegap synthesis(270 nucleotides)correspond to the primer of 40 and the product of 230 nucleotides.This band was quantifiedwith a PhosphorImager(MolecularDynamics) and documentedin Panel C. The data for pol 8 holoenzyme(PanelD) were quantifiedin the similarway and are thereforenot documentedas an auteradiogram.

ondary structures of M13 DNA, but on the other hand used less primers. The binding ofpol e to the primer recognition complex might decrease the turnover of the enzyme.

Cooperation o f pol tx/primase with pol 8 and pol e holoenzymes is influenced by RP-A and E.coli SSB Above we analyzed extension of preexisting synthetic DNA primers by both pol ~5and pole. Next we analyzed the cooperative action ofpol tx/primase with pol ~ or pol E on unprimed M13 DNA. Pol (z/primase itself under certain conditions is able to completely replicate M13 DNA

$138

Fig. 5. Replication of unprimed M13 DNA by calf thymus DNA polymerases o~ 5, ~ and combinations thereof in the presence of increasing amounts of E.coli SSB. Reaction mixtures conmined unprimed DNA, 0.25 U o f p e l ct (if added) and all the other components as indicated in Materials and methods. Samples were incubated 60 rain at 37~ and analyzed on a 1.5% alkaline agarose gel. The experimental design is outlined on the top of the autoradiogram.

by producing 3-10 product chains per circle (Nasheuer and Grosse 1987). On the other hand, PCNA/RF-C blocked pol ~ synthesis on RP-A covered ssDNA (Tsurimoto and Stillman 1991b). We analyzed DNA synthesis by pol r either alone or with pol ~ or pol e, in the presence of PCNA/RF-C and increasing concentration of either E.coli SSB (Fig. 5) or bovine RP-A (Fig. 6). A slight inhibition ofpol ~primase synthesis was observed in the presence of 300 ng of SSB in a mixture containing 200 ng ofunprimed M13 DNA. However, a very strong inhibition was observed when the amount of SSB in the reaction was rised to 600 ng or more. This concentration of SSB (300 ng per 100 ng of DNA) is in a good accord with optimal conditions for both pol ~ and e holoenzyme formation (Fig. 4, see also Podust et al. 1992). While synthesis by pol ~primase was strongly inhibited, a few primers could still be synthesized. Pol ~ and pol e efficiently utilized the few DNA primers synthesized by pol ~primase under these conditions and could synthesize long strands of DNA. In the absence of pol ~primase no products could be detected, confirming that the DNA preparation was essentially free from random primers (Fig. 7, lanes 13 and 14). Roughly similar results were obtained when homologous bovine RP-A was added to the reaction instead of heterologous E.coli SSB. A strong inhibition was detected when an amount 1.2-2 ~tg of RP-A was added per

200 ng of unprimed DNA. The same concentration of RP-A (600 ng per 100 ng of singly-primed M13 DNA) was found to be optimal for bovine pol ~ holoenzyme synthesis (data not shown). A comparison of Fig. 5 and Fig. 6 suggested that E.coli SSB was more effective for pol e and RP-A for pol 8 synthesis (Fig. 5, lanes 24, 25 for p o l e and Fig. 6, lanes 14,15 for pol 6). Thus the same conditions of primer recognition on one hand inhibited pol ~ from the action on ssDNA and favoured pol 6 and e utilization of 3'-OH termini.

Pol 5 and pol e holoenzymes can synthesize long DNA products on ssDNA ifpriming is carried out by pol ~primase Acting alone on unprimed DNA, pol ~primase synthesizes short products which can, upon longer incubation be repeatedly elongated ('Fig. 7, lanes 1-3). Under these conditions, we detected only short products synthesized by pol oJprimase after 10 min (Fig. 7, lane 1). Then PCNA, RF-C, SSB and pol ~5or pol e were added. The level of SSB was such that according to the data shown in Fig. 5 (lanes 9,10), further pol o~synthesis would be inhibited. Both pol 5 and pole holoenzymes synthesized long products comparable in size to the length of M13 DNA (Fig. 7, lanes 4--9). The simultaneous presence of both pol and e showed only additive effects (Fig. 7, lanes 8, 9, 12).

S139

Fig. 6. Replication of unprimed M13 DNA by calf thymus DNA potvmerases o~ 8, ~ and combinations thereof in the presence of increasing amounts of calf thymus RP-A. Reaction mixtures conmined unprimed DNA, 0.25 U ofpol ot (if added) and all the other components as indicated in Material and methods. Sampleswere incubated60 rain at 37~ and analyzed on a 1.5% alkaline agarose gel. The experimental design i s outlined on the top of the autoradiogram.

No difference in pol 8 and pol e products was evident when pol tx/primase preliminarily synthesized some primers or if it was supressed by addition of the auxiliary proteins (PCNA, RF-C and SSB) from the beginning of the reaction (Fig. 7, compare lanes 4-7 and 10, 11). Comparison of the efficiency of elongation of primers preannealed to M13 DNA and primers initiated by pol ix/ primase, by pol's fi and ~ holoenzymes indicated a significant difference between pol 8 and pol E. As we have described above, pol 8 displayed a higher dNMP incorporation than p o l e when a single primer was preannealed to DNA and equal amounts ofpol units (0.25 U) were used for both enzymes (compare lanes 8 and 9 to 11 and 12 in Fig. 3). On the other hand, p o l e was at least as efficient as pol 8 in the utilization the primers synthesized by pol ~primase (Fig. 7, lanes 4, 5 and 6, 7 or 10 and 11). In this case both longer products and a higher amount of dNMP incorporation could be detected for pole. Units for both pol ~ (in the presence of PCNA) and p o l e were determined on poly(dA)/oligo(dT) (Weiser et al. 1991) and quantitatively reflect the pol behaviour only on this template. To compare more carefully pol 5 and pol~ synthesis in different systems, we decreased the amount of pol added so that both pol 8 and pol e catalyzed the same nucleotide incorporation using primers preformed on M13 DNA. Under these conditions, pol ff./primase-dependent synthesis on M13 DNA, catalyzed by p o l e

Fig. 7. Comparison of DNA polymerase 8 and e holoenzymes in their ability to utilize the products synthesized by pol ot/primase on unprimed M13 DNA. Replication of unprimed M13 DNA was car-

ried out as describedin Material and methods and productswere analyzed on 1.5% alkaline agarose gel. Lanes 1, 2 and 3: Pol o/primase (0.4 U) synthesizedthe products in the absence of any auxiliary proteins for 10, 30 and 60 min respectively.Lanes 4 and 5:Pol o./pfimase initiated the DNA synthesisin the absenceof any auxiliary proteins for 10 min, then PCNA, RF-C, SSB and pol 8 (0.25 U) were added. Lanes 6 and 7:as lanes4 and 5 but pole (0.25 U) was added insteadof pol 8. Lanes 8 and 9: as lanes 4 and 5 but both pol 8 and pol Ewere added together. Additional incubationtime in the presence ofpol 8 or polc holoenzymeswere 30 min (lanes 4, 6, 8) or 60 min (lanes 5, 7, 9). Lanes 10-14: the auxiliary proteins PCNA, RF-C, SSB, pol ct and/or pol 8 and/or pol~ were all present from the beginning of the reaction and incubated for 60 rnin.

S140 holoenzyme, was 4.4-fold higher than that catalyzed by pol 8 (data not shown). Essentially the same result was obtained when 600 ng ofE.coli SSB protein was replaced in the reaction mixture by 1.2 I.tg of RP-A. In this case, pol e synthesized 2.8-fold more DNA product than pol 8 in the pol a-primase-dependent synthesis. Next the activity of pol 8 in the reaction was increased in order to achieve equal efficiency of utilization of the primers synthesized by pol tx/primase. Under these conditions, pol 8 holoenzyme incorporated 6-fold more DNA precursors in the preformed primers than pol e holoenzyme. The difference in the efficiency of utilization of these two types of primers on M13 DNA by both pol's may be partially explained from the different affinity (Kin) of the pol's for the primer termini (Fig. 2). However, a specific interaction of the pol e holoenzyme with pol tx/primase could also be suggested. SV40 replication studies have demonstrated that pol ~primase initiates the leading strand and then synthesis is switched to pol 8 (Tsurimoto and StiUman 1991b). The observation that pol 8 is more efficient on singly-primed M13 DNA than pol e is consistent with this idea. Nethanel and Kaufmann (1990) gave evidence that the lagging strand is synthesized by two pol's. Pol ~primase initiates the OkaTaki fragment but repeated elongation is prevented if PCNA, RF-C and RP-A are present. Therefore the data presented here indicate that pol e holoenzyme is better suited than pol 8 holoenzyme, to extend DNA synthesis initiated by pol tx/primase. Our data provide further evidence that pol s might be the second lagging strand pol as was originally suggested by Burgers (1991).

Conclusions The data presented in this paper indicate that: (i) Calf thymus pol 8 and pol e are both dependent on the three auxiliary proteins PCNA, RF-C and RP-A (SSB) for efficient DNA synthesis on singly-primed DNA, however, nature this dependence is different between pol 8 and pol e. (ii) Pol e holoenzyme synthesizes longer products, while at the same time, using less primers than pol e core. (iii) The cooperation ofpol tx/primase with pol 8 or pol e is influenced by RP-A and E.coli SSB and finally, (iv) both pol 8 and pol e holoenzymes can synthesize long products on M13 DNA if priming is carried out by pol ~primase. Our data support the idea that pol 8 is the leading and pol E is the second lagging strand replication enzyme.

Acknowledgements. This work was supported by the Swiss National Science Foundation (grants 31.28592.90 and 31.30298.90), by the Swiss Cancer Society and by the Kanton of Z~rich. The authors would like to thank Gerhard Cullman, Bettina Strack and Gregg Siegal for their suggestions on the manuscript.

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